Synthesis of amphiphilic semigrafted pseudo-Pluronics for self-assemblies carrying indomethacin

Abstract

Copolymers with semigrafted topology, consisting of a linear block of poly(ethylene glycol) (PEG) and polymethacrylic segments with loosely distributed oligo(propylene glycol) (OPG) grafts, were obtained by atom transfer radical polymerization (ATRP). The synthesis was performed with the use of a monofunctional macroinitiator (bromoester functionalized PEG) and OPG methacrylate macromonomer (OPGMA) with methyl methacrylate (MMA) comonomer. The amphiphilic copolymers with various compositions of hydrophobic grafted blocks were able to self-assemble in aqueous solution yielding particles with a variety of average sizes (105–210 nm determined by DLS), and the critical aggregation concentration (CAC = 0.032–0.086 mg mL⁻¹ determined by fluorescence spectroscopy). TEM images confirmed the formation of spherical micellar structures. Indomethacin (IMC), a poorly water-soluble drug, was selected as the model drug and encapsulated via the solvent evaporation method with evaluation by drug loading content (DLC = 15–90%). The in vitro release of IMC from polymeric particles in buffer solutions was pH-dependent (lower rates at pH = 5.0 than at pH = 7.4). The results indicated that the linear-b-graft copolymers may be potential carriers for delivery of poorly water-soluble drugs.

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Introduction

Pluronics are a family of block copolymers containing polyether segments, both poly(ethylene glycol) (PEG) and poly(propylene glycol) (PPG), which are mostly used as non-ionic surfactants because of amphiphilic properties, but they are also biocompatible and temperature-sensitive, providing medical applications.¹ The well-defined polymers, including stimuli-responsive macromolecules, prepared via controlled radical polymerizations (CRP) have great interest in science and industry, because of their unique properties.² The precisely designed macromolecular structures with proper functionalities, and compositions, as well as topologies, in combination with non-toxicity, and compatibility with human tissues, are great materials for biomedical applications, including drug delivery systems (DDS), tissue engineering and cell adhesion.³ The polymers in DDS are being designed to reduce the toxicity, increase the absorption, and improve the release profile of drugs.⁴ There are various types of DDS classified by a function of the structure and the release mechanism, e.g. membrane, matrix or micelle based systems and polymer–drug conjugates.⁵ Among the polymeric DDS systems the self-assembling structures, where the drug is entrapped by physical interactions, are particularly interesting and promising carriers due to its relatively small sizes, that enable them penetration through the small capillaries and taking up by the cells.⁶ The stability of the micelles is important parameter, which let to determine potential application in DDS.⁷

Pluronic-based micelles are characterized by unique core–shell architecture, which is suitable for encapsulation of drugs, transporting and releasing them in the human body. Additionally, their micellar cores are larger than the surfactant ones, generating capability for solubilization of hydrophobic drug in higher content. Thus, the Pluronics with flexible physicochemical properties were applied as micellar or hydrogel vehicles for various drugs in the treatment of tumors, including Doxorubicin^8,9 and Paclitaxel^10,11 or anti-inflammatory therapy, such as Ibuprofen¹² or Ketoprofen.¹³ Micelles based on amphiphilic block polymers, including Pluronics are most commonly used systems, but their stability is reduced in comparison to the analogs formed by non-linear polymers.¹⁴ Therefore, the common strategy affording the enhanced micelle stability in aqueous media is based on amphiphilic semigrafted,^15,16 grafted,^17,18 brush,^19,20 star^21,22 or dendrimer²³ structures.

In respect to this advantage, the standard Pluronics were reconstructed to the non-linear structures, such as P85 grafted on polyethyleneimine²⁴ and F127 on chitosan²⁵ to improve their properties (e.g. pH sensitivity, degradation). Alternatively, the oligoethers²⁶ have been modified to macromonomers, that is oligo(ethylene glycol) methacrylate with methoxy or hydroxy end group (mOEGMA or OEGMA, respectively), and hydroxyl functionalized oligo(propylene glycol) methacrylate (OPGMA) as the most common in design of graft copolymers, e.g. PPG-b-PEG grafted on polymethacrylate.²⁷ The use of the oligoether macromonomers let to generate branched copolymers demonstrating extraordinary properties. For example, copolymers of mOEGMA and biotin-3-aminopropyl methacrylamide were prepared for biospecific recognition,²⁸ whereas biodegradable and biocompatible naproxen-loaded micelles were obtained using brushes made of mOEGMA and cholesteryl-modified poly(L-lactic acid).²⁹ The miscellaneous topology of amphiphilic polymer brush-b-linear-b-brush used for formation of micelles with entrapped clofazimine, was obtained by poly(L-lactide) as bifunctional macroinitiator in the polymerization of OEGMA macromonomer.³⁰ In another case the graft copolymers of OPGMA and dimethylaminoethyl methacrylate yielded dual-responsive “reversible” micelles,³¹ which in aqueous media showed ability for co-delivery of paclitaxel and DNA to form a nano-sized polyplexes with excellent gene transfection efficiency.³² Copolymerization of mOEGMA, OPGMA, and ethylene glycol dimethacrylate resulted in photo-cross-linked hydrogels with the thermoresponsive swelling behavior.³³

In our studies we have explored new systems based on polymers with semigrafted topology (A-b-(B-graft-C)), which can be treated as non-linear pseudo-analogs of the Pluronics. They were prepared by hydrophilic monofunctional macroinitiator derived from poly(ethylene glycol) methyl ether (mPEG), which was used in the controlled radical copolymerization of hydrophobic OPGMA macromonomer and methyl methacrylate (MMA) (Scheme 1). In our previous work, the OEGMA and OPGMA have been applied in the synthesis of hydroxy-functionalized graft copolymers, in which oligoether side chains were extended with poly(methacrylic acid) segments^34,35 to investigate their drug carrier properties. In the current work the hydrophilic–hydrophobic balance in amphiphilic systems was adjusted by the length of grafted backbone and the grafting density of OPG side chains, whereas the length of hydrophilic linear PEG block was constant. The self-assemblies with encapsulated indomethacin (IMC) as the model anti-inflammatory drug were tested by the in vitro release experiments at acidic and neutral conditions to verify the usage properties as the future DDS. The advantages of the designed systems based on the non-linear pseudo-Pluronics are expected by the improved stability of polymeric particles with acceptable sizes and drug loading capacity for delivery over longer time than for micellar systems of standard Pluronics.

Scheme 1 Semigrafted pseudo-Pluronic type copolymers self-assembled with drug loading.

Results and discussion

Preparation of semigrafted copolymers

In the first step mPEG-based macroinitiator (mPEG–Br) was obtained by esterification of hydroxyl group to bromoester one in the presence of 2-bromoisobutyryl bromide and TEA. Next, the amphiphilic semigrafted mPEG-b-P(OPGMA-co-MMA) copolymers were synthesized by ATRP of OPGMA and MMA using mPEG–Br in the presence of CuBr/dNbPy or CuX/PMDETA (where X = Br, Cl) as catalytic systems (Table 1). The reactions were conducted with various initial content of OPGMA macromonomer (5–20 mol%) in anisole or anisole/methanol mixture at 60 °C or at the room temperature. In comparison to the previously synthesized graft copolymers P(OPGMA-co-MMA) with ethyl 2-bromoisobutyrate initiator,³⁵ the reaction rates of polymerizations initiated by mPEG–Br were significantly higher using the same conditions. This effect can be explained by more polar system supported with the hydrophilic fraction of PEG macroinitiator. The ¹H NMR analysis confirmed the chemical structures of the copolymers starting from mPEG modified to macroinitiator and the semigrafted copolymer (Fig. 1).

Table 1 Reaction conditions for copolymerization of MMA and OPGMA initiated by mPEG–Br at 60 °C

No.	[OPGMA]0/[MMA]0/[mPEG–Br]0	Catalyst system: [CuX]0/[ligand]0	Solvent
a 25 °C.
I	475/25/1	CuBr/dNbPy (0.75/1.5)	80 vol% (anisole)
II^a, III	475/25/1	CuBr/dNbPy (0.75/1.5)	30 vol% (anisole/methanol = 90/10 v/v)
IV	475/25/1	CuBr/PMDETA (1/1)	150 vol% (anisole)
V	475/25/1	CuBr/PMDETA (1/1)	100 vol% (anisole/methanol = 90/10 v/v)
VI	425/75/1	CuBr/dNbPy (0.75/1.5)	100 vol% (anisole/methanol = 90/10 v/v)
VII–VIII	425/75/1	CuCl/PMDETA (1/1)	30 vol% (anisole)
IX–XI	400/100/1	CuCl/PMDETA (1/1)	30 vol% (anisole)

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Fig. 1 ¹H NMR spectra of mPEG (a), bromoester-functionalized mPEG macroinitiator (b), and semigrafted mPEG-b-P(OPGMA-co-MMA) copolymer II (c).

The monomodal GPC traces of the semigrafted copolymers in Fig. 2 show a clear shift to lower elution volumes compared to that of mPEG–Br precursor, indicating an increase in the average molecular weights (Table 2). Generally, the copolymers I–VIII (5–15 mol% of the initial macromonomer amounts) were characterized by narrow molecular distributions (M_w/M_n = 1.2–1.4), suggesting that the polymerizations were better controlled than IX–XI synthesized with 20 mol% of OPGMA (M_w/M_n > 1.45).

Fig. 2 GPC traces of mPEG and semigrafted copolymers with various content of OPGMA units.

Table 2 Characteristics of the synthesized semigrafted copolymers of mPEG-b-P(OPGMA-co-MMA)

No.	OPGMA initial content [mol%]	Time [h]	NMR			GPC		Water solubility
			Conversion [%]	DPn	PEG fraction [mol%]	Mn [g mol^−1]	Mw/Mn
I	5	2	19.3	97	53.9	12 900	1.25	Yes
II	5	2	14.7	74	60.5	10 500	1.19	Yes
III	5	3	18.9	94	54.5	6800	1.32	Yes
IV	5	0.6	24.4	122	48.1	8800	1.39	Yes
V	5	0.16	40.5	202	35.1	15 400	1.25	Yes
VI	15	2	28.9	144	43.9	43 200	1.20	Yes
VII	15	1.5	34.8	174	39.4	35 600	1.40	Yes
VIII	15	3	59.9	299	27.4	38 900	1.35	No
IX	20	0.5	50.1	250	31.1	21 800	1.47	No
X	20	1	72.1	360	23.9	26 000	1.60	No
XI	20	2	77.0	385	22.7	29 900	1.62	No

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The short polymethacrylate segments of backbone (<100 repeating units) were obtained in the case of copolymers I–III, which were prepared at 5 mol% of OPGMA with CuBr/dNbpy as the catalyst system. The use of higher activity catalysts, that is copper halide complexed by PMDETA, afforded the improvement of polymerization degree. For instance, at the same initial ratio of methacrylates, higher conversion was yielded within 10 minutes in the less diluted system V in comparison to IV, resulting in polymethacrylate with ∼200 repeating units. Similar lengths of polymethacrylic chains were also attained increasing the initial amount of OPGMA up to 15 mol% (VI, VII), where the extension of reaction time let to form twice longer polymethacrylate chain (VII vs. VIII). Copolymers IX–XI (20 mol% of OPGMA) containing 250–400 units in polymethacrylate segment were resulted under the same conditions as for VII–VIII. Because of the constant length of mPEG block in the semigraft copolymers, the longer P(OPGMA-co-MMA) chains (VIII–XI) were related to larger amounts of hydrophobic fraction (>70 mol%), which significantly limited polymer solubility in water and their availability for self-assembly studies in aqueous solution.

Micellar systems and drug loading characterization

In aqueous media the semigrafted copolymers with amphiphilic properties were self-organized into micellar aggregates. This specific architecture of copolymers, which can be treated as non-linear triblock copolymers combining linear hydrophilic and grafted hydrophobic segments, let to assume formation of core–shell particles, in which polymethacrylic backbone is placed in the core together with the OPG grafts, whereas the well-soluble in water PEG segments are stretched to supply shell (Scheme 1). The TEM observations confirmed the formation of particles with regular spherical shapes and sizes ranged in 100–200 nm as it is presented in Fig. 3 for the representative copolymer V.

Fig. 3 TEM images of particles formed by the semigrafted mPEG-b-P(OPGMA-co-MMA) copolymer IV at concentration of 1 wt% in deionized water.

The aggregation of mPEG-b-P(OPGMA-co-MMA) copolymers was also evidenced by detection of CAC using the fluorescence technique with pyrene as a probe. As it is shown in Fig. 4, the intensity ratio of I₃₃₈/I₃₃₃ was changed sharply above the CMC value that indicates the presence of pyrene entrapped into particle cores. The CAC of the studied systems were determined to be in the range of 0.032–0.086 mg mL⁻¹ (Table 3). These relatively low values suggest that the particles formed from mPEG-b-P(OPGMA-co-MMA) can remain stable in solution even after high dilution. In the linear block copolymers this parameter is corresponded primarily with molecular weight and hydrophobicity of the micellar core. In our study the dependence of CAC on molar content of hydrophobic fraction (Fig. 5) indicated increasing tendency for copolymers with DP_n above 95 with exception of IV, but in this case dispersity was the highest in the group of water soluble semigraft copolymers.

Fig. 4 Plot of the intensity ratio I₃₃₈/I₃₃₃ from pyrene excitation spectra (at λ = 390 nm) versus nanoparticle concentration (log C) for mPEG-b-P(OPGMA-co-MMA) copolymer I.

Table 3 Characteristics of polymeric particles, drug loading and IMC-released data at various pH conditions

	Hydrophobic fraction (MMA + OPGMA) [mol%]	CAC [mg mL^−1]	Blank particles		IMC-loaded particles		DLC [wt%]	IMC released after 72 h [%]
			Dh [nm]	PDI	Dh [nm]	PDI		pH 5.0	pH 7.4
I	0.461	0.086	106	0.354	145	0.285	22.3	30	69
II	0.395	0.040	144	0.407	177	0.416	14.1	25	38
III	0.465	0.032	181	0.360	194	0.412	87.7	33	54
IV	0.519	0.036	118	0.447	129	0.455	18.5	18	28
V	0.649	0.036	210	0.379	235	0.426	64.2	13	23
VI	0.561	0.056	164	0.240	183	0.332	62.7	16	26

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Fig. 5 Dependence of hydrodynamic diameter (left Y axis) and CMC (right Y axis) on polymerization degree of the hydrophobic grafted segment.

In comparison with the literature data for the critical concentrations to form the self-assemblies of Pluronics, for example F127 (2.5 g dl⁻¹),³⁶ P103, P123, F127 (2.5–3.5 g L⁻¹),³⁷ or linear Pluronic-based polymers, F127 and P85 modified by poly(lactic acid) and folic acid (1.4 and 1.9 mg L⁻¹),³⁸ the CAC's of the synthesized semigrafted copolymers are lower, suggesting higher stability of the formed self-assemblies, which could be used as the drug carriers with perspectives for extended time of drug release.

The poorly soluble in water IMC, that is a member of non-steroidal anti-inflammatory drugs used to reduce pain in rheumatoid arthritis, osteoarthritis, headache and so on, was selected for encapsulation into the hydrophobic particle core. We have verified amphiphilic copolymers with semigrafted topology as a drug carrier systems because of its promising abilities for increasing the IMC dissolution rate as well as preventing the relatively high IMC permeability. Thus, we were able to obtain micellar carriers with potential high bioavailability into the body. IMC was loaded into the polymeric particles by physical interactions using solvent evaporation method. The drug loading content (DLC) as well as the hydrodynamic diameters without and with the loaded drug (D_h) are presented in Table 3. Sizes of the blank self-assemblies were in the range of 106–209 nm with narrow size distributions (0.24–0.45). The particles increased proportionally to the length of hydrophobic polymethacrylic block above DP_n = 95 (Fig. 5). IMC entrapping did not noticeably affect particle sizes, which increased by about 20 nm reaching values in the range of 130–235 nm with similar dependency on hydrophobic fraction as in the case of blank particles (Fig. 5 and 6). Since, the size of micellar structures is one of the important parameters to qualify them as potential drug carriers, the most efficient systems are correlated with the sizes up to 200 nm. In almost all cases the obtained drug loaded particles displayed suitable sizes, which are required for the drug delivery.

Fig. 6 Size distribution profiles (by intensity) of micelles based on mPEG-b-P(OPGMA-co-MMA) copolymer (IV) measured by DLS at 25 °C in aqueous solution of blank micelles (a), and IMC-loaded micelles (b).

Generally, two trends of drug loading dependent on particle sizes were distinguished, that is smaller ones below 150 nm (I–II, IV) with lower DLC values (14–22 wt%) vs. bigger ones above 150 nm (III, V–VI) with significantly higher loading capacity (63–88 wt%). The comparable IMC loading contents were already reported for the other types of grafted polyether-based systems, that is hydroxyl-functionalized PEG graft copolymers (22–88%).³⁹

In vitro release profiles of IMC from polymeric particles

To evaluate the effects of both pH and composition dependence on drug release properties of the IMC-loaded particles, in vitro drug release studies were performed in acetate buffer (pH 5.0) and phosphate buffer solution (pH 7.4) at 37 °C. These buffers were selected to simulate physiological pH of the gastrointestinal fluid (pH ∼ 4.8), and the small intestine or colon (pH = 7.5 ± 0.4 or 6.4 ± 0.6), as well as the bloodstream (pH = 7.4 ± 0.5). As it was shown by data summarized in Table 3, the pH value of the medium had strong effect on the release rate of IMC from mPEG-b-P(OPGMA-co-MMA) particles. In each studied micellar system, the release rate of IMC was much higher at pH 7.4 than at pH 5.0 (Fig. 7a).

Fig. 7 Comparison of IMC release profiles from micellar systems at pH 7.4 vs. 5.0 (a) and correlation between amount of drug release and content of hydrophobic fraction at pH = 7.4 (b).

Additionally, the release profiles exhibited initial burst, followed by a slow release in all polymeric systems. The influence of the hydrophobic content on release rate was also observed. The copolymer V with the longer polymethacrylic backbone (DP_n = 202; 65% of hydrophobic fraction) exhibited slower release profile than polymer I with twice shorter graft segment (DP_n = 97; 46% of hydrophobic fraction). For example 9% of IMC at pH 5.0 and 13% at pH 7.4 was released within 24 h from copolymer V, whereas the release rate of copolymer I reached values 16% at pH 5.0 and 59% at pH 7.4 (Fig. 8a). In the case of copolymers differing in the DG some changes in releasing rate were also seen, that confirm the influence of composition on diffusion rate. Comparing the release profiles of samples I and VI with different DG, but similar DP_n (97 vs. 144, respectively), slower release of IMC for copolymer VI than I (9% vs. 16% at pH 5.0 and 16% vs. 59% at pH 7.4 after 24 h) was detected (Fig. 8b). It is worth to notice that the largest amounts of the released drug (I and III) are correlated to extreme values of CAC (0.086 and 0.032 mg mL⁻¹) for the studied self-assembly systems (Fig. 7b).

Fig. 8 Zero-order plot of percentage of IMC released in time from selected micelles varied with length of polymethacrylate segment (a) or grafting density of OPG side chains (b) in different pH 7.4 and 5.0 at 37 °C.

The release behavior is well fitted to the first-order kinetic model expressed by semilogarithmic plot of percentage of drug remained inside the particle vs. time with the correlation coefficient R² = 0.9–0.98 (Fig. 9a). The kinetic analysis revealed that the release of IMC by the mPEG-b-P(OPGMA-co-MMA) based particles was well fitted by Higuchi model (R² = 0.80–0.96), suggesting superiority of diffusion process (Fig. 9b).

Fig. 9 The kinetics of IMC release at pH 7.4 and 5.0 at 37 °C from polymeric micelles as linear plot of log(% remaining drug) vs. time in accordance with the first-order equation (a), linear plot of % of released drug vs. square root of time in accordance with the Higuchi model (b).

The releasing profiles of IMC encapsulated by the semigrafted pseudo-Pluronics indicated that these drug systems can be recommended for oral products, which in the stomach at acidic conditions are able to provide releasing in relatively longer time in comparison to the neutral bloodstream environment. The rectal administration characterized by a faster onset and shorter duration than the oral route is also plausible, though the drug system would be intensively adsorbed and distributed by blood vessels.

Experimental

Materials

Poly(ethylene glycol) methyl ether (mPEG, Aldrich, 5000 g mol⁻¹), anisole (Fluka, 99%), heptane (POCH), toluene (POCH) were dried over molecular sieves and stored in a freezer under nitrogen, whereas oligo(propylene glycol) methacrylate (OPGMA, Aldrich, 375 g mol⁻¹, n = 5, ESI: Fig. S1†), methyl methacrylate (MMA, Aldrich, 99%) were purified from inhibitors by passing through the alumina column or distillation, respectively. Cooper(I) bromide (CuBr, Fluka 98%) and copper chloride(I) (CuCl, Fluka 97%) were purified by stirring with glacial acetic acid, followed by filtration and washing the solid with ethanol (three times) and diethyl ether (two times). Then, the CuBr and CuCl were dried under vacuum for 2 days. Ion-Exchange Resin Dowex® Marathon™ MSC hydrogen form (Aldrich) was activated by stirring with nitric acid and water, than dried at 50 °C. 4,4′-Dinonyl-2,2′-dipyridyl (dNdpy, Aldrich, 97%), N,N,N′,N′′,N′′-pentamethyldi-ethylenetriamine (PMDETA, Aldrich, 99%), triethylamine (TEA, Sigma-Aldrich ≥99%), α-bromoisobutyryl bromide (BriBuBr, Sigma-Aldrich), 2-{1-[(4-chlorophenyl)carbonyl]-5-methoxy-2-methyl-1H-indol-3-yl}acetic acid known as indomethacin (IMC, Alfa Aesar, 98%), were used as received. All other solvents were applied without purification.

Preparation of PEG-based macroinitiator (mPEG–Br)

mPEG (5 g, 1.0 mmol) and triethylamine (0.13 g, 1.3 mmol) in dry toluene (50 mL) were placed in a round-bottom flask. The solution was cooled with an ice bath and 2-bromoisobutyryl bromide (0.36 g, 1.2 mmol) was slowly dropped. The mixture was filled with nitrogen for 2 h at 0 °C, sealed and left under stirring for 24 h at room temperature. The precipitated insoluble salt was removed by filtration and using the DOWEX Ion Exchange resin. The filtrate was evaporated in vacuum. The purification based on extraction by 1 M HCl (30 mL) with CH₂Cl₂ (3 × 20 mL), washing with water (50 mL), saturation with Na₂CO₃ (30 mL), drying with MgSO₄, filtration, concentration under reduced pressure, and recrystalization from ethanol and petrol ether, yielded 4.75 g of macroinitiator (yield = 95%). GPC (THF): M_n = 7600 g mol⁻¹, M_w/M_n = 1.08. ¹H-NMR (δ, ppm, 300 MHz, CDCl₃): 1.94 (s, 6H, C(CH₃)₂Br), 3.38 (s, 3H, OCH₃), 3.42 (t, 2H, OCH₂), 3.64 (br s, OCH₂CH₂O), 3.87 (t, OCH₂), 4.33 (t, 2H, COOCH₂); M_n = 4480 g mol⁻¹, n = 98.

Synthesis of semigrafted copolymers (example for IV)

The comonomers OPGMA (0.22 mL, 0.59 mmol) and MMA (1.2 mL, 11.21 mmol), mPEG–Br (0.125 g), anisole (2.15 mL), PMDETA (4.9 mL, 0.02 mmol) were added to a Schlenk flask with a magnetic stirring bar, and degassed by three freeze–pump–thaw cycles. Then CuBr (3.38 mg, 0.02 mmol) was introduced to the reaction mixture and an initial sample was taken. The stirred flask was immersed in an oil bath thermostated at 60 °C to start the reaction. The polymerization was stopped by exposing the reaction mixture to air, and after that it was diluted with chloroform (CHCl₃), passed through an alumina column to remove the catalyst and concentrated by a rotary evaporator. The polymer was isolated by precipitation into cold heptane, filtered, and then dried under vacuum to a constant mass. ¹H-NMR (δ, ppm, 300 MHz, CDCl₃): 0.7–1.3 (m, 3H, CH₃ in polymethacrylic backbone and in OPG side chains), 1.6–2.2 (m, 2H, CH₂ in polymethacrylic backbone), 3.6 (s, 3H, OCH₃), 3.4–4.4 (m, 2H + 1H, OCH₂ and OCH).

Preparation of polymeric particles by the solvent evaporation

The amphiphilic copolymers (without/with drug) were first dissolved in CH₂Cl₂, then deionized water (around 2 fold excess relative to CH₂Cl₂) was added dropwise under gentle stirring and the systems were stirred for 24 h. Next, the CH₂Cl₂ was slowly evaporated under the air and the resulting aqueous solutions were lyophilized. In the case of drug loaded particles before lyophilization the unloaded drug was separated by sonication and centrifugation. The weight ratio of polymer : drug was 1 : 1.

The amount of encapsulated IMC was quantitatively determined by a UV-vis technique. The calibration curve used for drug loading characterization was established by the intensity of IMC with different concentrations in THF. Drug loading content (DLC) was calculated from the following equation:

In vitro drug release study and release kinetics

IMC release from polymeric micelles was determined at 37 °C in acetate buffer (pH = 5.0) and phosphate buffer (pH = 7.4). After lyophilization, the solution of drug-loaded micelles dissolved in buffer (2 mg mL⁻¹) was transferred into dialysis cellulose tube (MWCO 3.5 kDa membrane, Spectrum Laboratories Inc.), which was immersed into 20 mL of corresponding buffer in a glass cylinder and stirred at 37 °C for several days. At appropriate time intervals, 2.5 mL of the buffer solution sample from the released medium in outside of membrane was taken out for measurements. The concentration of released drug was estimated by UV-vis spectroscopy at λ = 320.5 nm. Each result is an average of three parallel measurements.

Characterization

Molecular weights and their distributions (M_n, M_w/M_n) were determined by gel permeation chromatography (GPC) equipped with an 1100 Agilent isocratic pump, autosampler, degasser, thermostatic box for columns, and differential refractometer MDS RI Detector. Addon Rev. B.01.02 data analysis software (Agilent Technologies) was used for data collecting and processing. Pre-column guard 5 mm (50 × 7.5 mm) and PLGel 5 mm MIXED-C (300 × 7.5 mm) column were used for separation. The measurements were carried out in THF as the solvent at 30 °C with flow rate of 0.8 mL min⁻¹ and using calibration based on a polystyrene standards.

¹H nuclear magnetic resonance (NMR) spectra for structure analysis were recorded on UNITY/INOVA (Varian) 300 MHz spectrometer using CDCl₃ as solvent and tetramethylsilane as an internal standard. The monomer conversion determined by ¹H NMR was used to calculate the polymerization degree of methacrylate segment in the backbone (DP_n, Table 1) by following equation: DP_n = conversion × [PPGMA + MMA]₀/[mPEG–Br]₀.

The critical aggregation concentration (CAC) of micelles was measured by fluorescence spectrophotometry (Hitachi F-2500) using pyrene as fluorescence probe. Excitation spectra of pyrene (λ_em = 390 nm) were recorded at polymer concentrations ranging from 2 × 10⁻³ to 1 mg mL⁻¹ and constant concentration of pyrene (3 × 10⁻⁴ mM). The intensity ratios of I₃₃₈ to I₃₃₃ were plotted against the log of concentration of the copolymer solutions.

The IMC loaded content and its release profiles were evaluated using Spectrophotometer UV/VIS Nicolet Evolution 300.

Sizes of particles and their polydispersities (PDI) were determined by dynamic light scattering (DLS). Measurements were carried out in 25 °C using a Malvern Zetasizer Nano-S90 equipped with an 4 mW He–Ne ion laser operating at λ = 633 nm. All of the sample measurements were performed at a fixed scattering angle of 90°. At least 4 correlation functions were analyzed per sample in order to obtain an average value. The concentration of samples in deionized water was 1 mg mL⁻¹.

Morphologies of particles were investigated by transmission electron microscopy (TEM) using 120 kV FEI Tecnai G2 Spirit BioTWIN instrument. Measurements were conducted for polymer samples at concentration of 1 wt% in deionized water.

Conclusions

The amphiphilic mPEG-b-P(OPGMA-co-MMA) copolymers with semi-grafted topology were investigated as potential carriers of IMC. These macromolecules displayed self-assembly behavior in water to form polymeric nanoparticles at relatively low CAC values, which suggest suitable physical stability of the micellar structures. They also demonstrated varied ability of IMC loading (<25 and >60% of drug content), which was correlated with the sizes of blank superstructures (below and above 150 nm, respectively). In vitro drug release profiles showed much faster rate of drug releasing at pH 7.4 in comparison to pH 5.0. The influence of polymer structural parameters on micellization/aggregation, particle size, drug loading content and release rate was verified yielding variety of behaviors. The results of drug release kinetics are well expressed by the Higuchi model, that confirms the occurrence of diffusion process in IMC releasing. The results also confirm that the prepared polymeric systems can be applied as drug carriers for other poorly water-soluble drugs, including the anti-inflammatory drugs.

Acknowledgements

P. M. is a scholar under the project “DoktoRIS” co-financed by European Union under the European Social Fund. The authors thank Professor A. Dworak and MSc. K. Łaba for possibility to use the fluorescence spectrophotometer in the Centre of Polymer and Carbon Materials (Polish Acad. Sci.). Many thanks for Dr A. Mielańczyk for GPC analysis (Silesian University of Technology), and MSc. Ł. Mielańczyk for TEM images (Medical University of Silesia).

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Footnote

† Electronic supplementary information (ESI) available: ¹H NMR spectrum of OPGMA macromonomer. See DOI: 10.1039/c6ra20368j

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